1. Field of the Invention
The present invention relates generally to enzyme immobilized membranes and a method for producing the same, and more specifically, to a semiconductor enzyme sensor utilizing such enzyme immobilized membranes.
2. Description of the Prior Art
In the past, enzymes have been industrially utilized as catalysts, particularly in the fermentation industry, and the like. In general, enzymes were dissolved or dispersed in aqueous solutions for promoting various chemical reactions and, after completion of the reactions, the enzymes were not recovered from the solutions but discarded uselessly each time the respective reactions had been finished. In recent years, however, enzyme immobilization techniques have been developed which enable repeated use of enzyme in a stable state, whereby areas of utilization of enzymes have been rapidly expanded, for example, to a field of measuring various parameters of chemical substances in addition to traditional fields of fermentation, syntheses of chemical substances, and the like.
The measurements of concentrations of respective components contained in body fluids such as blood or urine are very important for clinical diagnosis and thus there have been developments and/or improvements made of various kinds of quantitative measurements. Of these, many enzyme sensors have been proposed which are able to effect quick and continuous measurements by employing enzyme immobilized membranes and varying kinds of electrodes.
Such enzyme sensors have been studied since about 1970 and today it becomes possible to measure chemical and physical quantities of a wide variety of substances by the use of enzyme sensors.
In the enzyme sensors fabricated in the early years, an enzyme immobilized membrane was physically or chemically adhered to a sensitive portion of an enzyme sensor which is adapted to convert physical or chemical quantities such as temperature, ion concentration, gas concentration or the like into electrical signals. In these days, however, in accordance with miniaturization and/or multiplication of enzyme sensors, it becomes necessary to selectively form an enzyme immobilized membrane on a limited area of a sensitive portion of a sensor.
In order to measure chemical quantities of a substance, an enzyme immobilized membrane is suitable which has a thin thickness and a limited area.
Biosensors are typical examples of sensors utilizing enzyme immobilized membranes for measurements of chemical substances. Such a biosensor comprises an enzyme immobilized membrane, and a transducer adapted to detect substances consumed or produced in the membrane and generate electrical signals upon detection of such substances. In this case, the enzyme immobilized membrane serves to discriminate a specific chemical substance to be measured, and cause a change in quantity of a material which corresponds to a change in the chemical substance and which is able to be detected by the transducer.
Among such biosensors, there are known those which employ a combination of a glucose immobilized membrane and an oxygen electrode, a combination of a glucoseoxidase immobilized membrane and a hydrogen peroxide electrode, a combination of glucoseoxidase immobilized membrane and a pH electrode, or the like. Glucose oxidase acts to decompose the glucose in the presence of oxygen in an enzyme immobilized membrane into gluconic acid and hydrogen peroxide according to the following reaction equation: EQU glucose+O.sub.2 .revreaction.D-glucono-.delta.-lactone+H.sub.2 O.sub.2 .dwnarw..uparw.H.sub.2 O gluconic acid
Accordingly, it is possible to measure the concentration of glucose by detecting the quantity of oxygen consumed in the above reaction with the oxygen electrode, the quantity of hydrogen peroxide produced in the above reaction with the hydrogen peroxide electrode, and a reduction of pH due to production of gluconic acid with the pH electrode.
Further, in order to form an enzyme immobilized membrane as employed in a miniaturized or multiplicated enzyme sensor as referred to above, a water soluble photosensitive resin such as, for example, photosensitive polyvinyl alcohol (PVA) is subject to a photolithographic process. FIGS. 1A through 1C show a series of procedures in a conventional method for forming an enzyme immobilized membrane from a photosensitive resin by means of photolithography. In these figures, reference numeral 1 designates a base or substrate such as, for example, a silicon wafer having a thermally grown oxidized film or membrane, a base board having amino groups introduced therein through a silane coupling agent, a transducer, a semiconductor sensor or the like. Provided on the entire surface of the substrate 1 by spin coating is an enzyme membrane 3 formed of a water soluble photosensitive resin containing an enzyme 2 such as glucose oxidase dissolved therein, as illustrated in FIG. 1A. Then, the enzyme membrane 3 thus formed is irradiated by ultraviolet light or visible light 6 through an appropriate photomask or filter 5 so that only a specific portion 4 of the enzyme membrane 3 or a sensitive portion 4 of the substrate 1 is subject to the irradiation of the light to provide photo crosslinking therein (see FIG. 1B). Thereafter, the remaining portion of the enzyme membrane 3, not irradiated by the light 6 and having no photo crosslinking, is developed and washed with water (see FIG. 1C).
With the above-described conventional method for forming an enzyme immobilized membrane, there arise the following problems due to the water development carried out after formation of photo crosslinking. Specifically, the specific portion having photo crosslinking is liable to be peeled off during the water development, and does not have sufficient mechanical strength for use in an aqueous solution; and the enzyme immobilized in the enzyme membrane 3 is liable to elute therefrom. Such phenomena become more remarkable as the amount of enzyme in the enzyme membrane 3 increases.
As a result, it is difficult to produce a highly sensitive biosensor in accordance with the conventional method for forming an enzyme immobilized membrane.
In general, there is another conventional method for making an enzyme immobilized membrane in an enzyme sensor for measuring glucose in which crosslinking is formed between glucose oxidase and bovine serum albumin by means of glutaraldehyde. FIG. 2 is an imaginary view showing a glucose-oxidase immobilized membrane formed on a base or transducer 1 having amino groups introduced thereinto by a silane coupling agent.
In the above glucose oxidase immobilized membrane forming method, an enzyme 2 in the form of glucose oxidase and bovine serum albumin 7 are dissolved in an aqueous solution, and then mixed with a solution containing an appropriate concentration of glutaraldehyde 8. The mixture thus formed is coated on the base 1 and kept in this state so that chemical crosslinking is produced between enzyme 2-enzyme 2, enzyme 2-bovine serum albumin 7, bovine serum albumin 7- bovine serum albumin 7, and enzyme 2-amino groups on the surface of the base 1, bovine serum albumin 7- amino groups on the base surface under the action of glutaraldehyde. In this manner, the enzyme 2 is captured in a three-dimensional polymer membrane, as illustrated in FIG. 2, and thus becomes insoluble in water and immobilized.
With recent enzyme sensors, there is a strong need for miniaturization and hence finely machinable materials and production methods therefore are required for such glucose oxidase immobilized membranes. The conventional glucose oxidase immobilized membranes are produced from the above-mentioned chemically crosslinked materials in accordance with the above bovine serum albumin-glutaraldehyde method, but involve a problem in that it is impossible to form enzyme immobilized membranes having limited areas with good reproducibility.